Objective To investigate the optimal b value in diffusion weighted MR imaging for diagnosis of prostate cancer at 3-T while using magnetic resonance/transrectal ultrasound (MR/TRUS) fusion-guided biopsy and system biopsy pathological findings as reference standard. Methods Forty-three consecutive patients were enrolled in this prospective study; they were suspected to have prostate cancer and underwent T₂WI and diffusion-weighted MRI with 10 b-values (0,50,100,150,200,500,800,1 000,1 500,and 2 000 s/mm²) prior to biopsy. DWI combined with T₂WI analysis was used for detecting prostate cancer. The biopsy modality was decided by the MRI results: patients had suspicious lesions took MR/TRUS fusion-guided biopsy and system biopsy and those had no suspicious lesions took system biopsy only. The signal intensity of prostate cancer lesions and normal prostate tissue on DWI of different b values were measured. Statistical analyses were performed by a Wilcoxon Signed Ranks test to compare the signal intensities between prostate cancer and normal prostate tissue. Receiver operating characteristics (ROC) were used to calculate the AUC, sensitivity, and specificity, so as to determine the optimal b value for revealing prostate cancer on DWI. Results Twenty-two of the 43 patients were diagnosed as prostate cancer, with 16 diagnosed as benign prostate hyperplasia (BPH) and 5 as prostatitis. Among the 22 prostate cancer patients b=1 500 s/mm² and b=2 000 s/mm² DWI images provided the best lesion visibility in 16/22 and in 6/22 patients, respectively. Wilcoxon Signed Ranks test showed that the signal intensities of the lesions and the normal tissues were significantly different(P<0.000 1) at all b values but when at 500 s/mm² (P=0.236). ROC curve indicated that the optimal signal intensity contrast was obtained at b=1 500 s/mm² (P<0.000 1, AUC=0.933), and the signal intensity cut-off value was 49.2, with the sensitivity and specificity being 0.909 and 0.909, respectively. Conclusion A b value of 1 500 s/mm² is optimal for 3-T DWI to visualize prostate cancer lesions, and b=500 s/mm² has the lowest diagnosis efficiency.